PROJECT SUMMARY Metabolic reprogramming is a hallmark of cancer that supports the rapid proliferation and survival of tumor cells. While many studies have focused on identifying pathways involved in increased glucose uptake and metabolism by tumor cells, many cancers (particularly acute leukemias) do not depend on glucose and instead prefer to metabolize fats to support their survival and growth. Despite the pervasiveness of this phenotype, molecular mechanisms that regulate fatty acid oxidation (FAO) in cancer remain largely unknown. As pathways that drive fuel addiction may provide new therapeutic targets or biomarkers for personalized therapy, there is a critical need to identify pathways that regulate dependency on lipids. We have discovered a new nutrient- dependent signaling pathway that controls fat oxidation in cancers via a little studied member of the prolyl hydroxylase domain protein family, PHD3. PHDs are a family of ?-ketoglutarate dependent dioxygenases that hydroxylate substrate proline residues and have been linked to fuel switching in cancer. We find that PHD3 regulates fatty acid metabolism by hydroxylating acetyl-CoA carboxylase (ACC2), a regulator of mitochondrial FAO. In response to nutrient abundance, PHD3 activates ACC2 to inhibit catabolism of fatty acids. Our proposal will test the hypothesis that tumors with low PHD3 will have excessive fatty acid oxidation due to deregulation of ACC2 activity, and that PHD3 levels may provide a new metabolic biomarker to identify tumors vulnerable to therapies that target fat catabolism. This proposal will examine the mechanism by which PHD3- mediated hydroxylation results in the specific activation of the ACC2 isoform (Aim 1). We will also examine the physiological stimulation of PHD3 under high nutrient conditions, and examine its coordination with AMPK signaling, which represses ACC by phosphorylation (Aim 2). Finally, we will examine the consequences of PHD3 activity, ACC2 hydroxylation, and FAO in AML survival and growth by examining the effects of PHD3 overexpression and vulnerability of tumors with low PHD3 to fat oxidation inhibitors (Aim 3). Our overarching goal is to elucidate the elements of PHD3 signaling and to leverage these findings to develop therapeutic strategies to treat tumors dependent on fat oxidation.